Proteomics of post-translational and chemical modifications of proteins and interactions of proteins of importance in food allergy

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

Hormones of thyroid gland in sera of rats treated with different dose of concentrated potassium iodine solutions

Introduction Potassium iodine (KI) is used as a drug therapy for treating numerous diseases such as small-vessel vasculitis, erythema nodosum, vasculitis nodularis, Sweet's syndrome, tuberculosis and granulomatosis, and for iodized salt. At the same time, KI can be harmful. Iodine intake may increase the frequency of thyroiditis in humans, and may induce the occurrence of experimental thyroiditis (ET) in animals. Investigations on an experimental model for the examination of thyroiditis in Wistar rats have clearly showed morphological changes in the rat thyroid evoked by KI administration. Objective The purpose of this study was to compare the effects of low and high doses of KI on the thyroid gland of Wistar rats and determine the effect on hormone status (T4, T3 and TSH) in this rat strain. Methods Two groups of rats from the Wistar strain were treated with a low iodine dose (225 μg/g BW) and with a high iodine dose (675 μg/g BW) of KI solutions. Untreated nonimmunized animals served as controls. The solution was administrated daily intraperitoneally during the period of 26 consecutive days. Results Monitoring hormone status (TSH, T3 and T4) and morphological changes it was found that therapeutic doses of KI applied in treatment induced the occurrence of experimental thyroiditis (chronic destructive Hashimoto's thyroiditis in humans) and cell necrosis in animals not carrying a genetic susceptibility. Significant inflammatory changes were observed in rats treated with a high iodine dose. Conclusion The early iodine induced cell necrosis and inflammation in the nonimmunized animals without genetic susceptibility is a new experimental model of thyroiditis.

Diffusion weighted imaging and magnetic resonance-spectroscopy in differentation of brain tumors

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..

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

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

A survey of Paleolithic sites in the Western Morava Valley

First evidence of habitation from Lower and Middle Palaeolithic in the Zapadna (Western) Morava valley were discovered few years ago. Sites located at the highest river terraces near Čačak (Kosovska Kosa, Viljuša, Vojnovića Brdo) yielded large quantity of artifacts of Lower Palaeolithic type including choppers, points of Quinson type and denticulated and notched tools on asymmetrical lakes. Similar situation has been encountered at Samaila-Vlaška Glava near Kraljevo, where Kombewa, Levallois and „proto-Levallois“ cores were discovered together with choppers as well as various types of sidescrapers and denticulated and notched tools. It has been established in the course of more recent site surveying that Palaeolithic inds also appear on the slopes of the Goč Mountain in the vicinity of Vrnjačka Banja. Artifacts have been encountered in the vicinity of secondary deposits of chert, opal and chalcedony in the area of villages Rudjinci and Štulac. Two partially worked bifaces were discovered at the site Rudno Brdo near the village Štulac, while tools made on large lakes (cleaver-like bifaces, massive points , notched tools) and on lakes some of which have the Levallois morphology were found on the banks of the Crnobarac creek. Middle Palaeolithic artifacts have been discovered also at lower terraces in the vicinity of Vrnjačka Banja (Ruđnci) and Kruševac (Globoder). Despite the fact that it concerns the surface scatters, whose integrity has yet to be conirmed, spatial distribution and technological variability of assemblages suggest an assumption that Western Morava valley was densely populated in the earliest phases of the Palaeolithic.У: Антоновић, Д., Филиповић, В. (ур.), XXXVIII Скупштина и годишњи скуп САД: програм, извештаји и апстракти, 04-06. јун 2015, Пирот, Србија (стр. 79). Београд и Пирот: Српско археолошко друштво; Музеј понишавља Пирот. (ISBN 978-86-80094-01-4) Теренска истраживања су финансирали Ерик Хефтер са Универзитета Аризона, Народни музеј у Краљеву и Културни центар у Врњачкој Бањи

Miokardni mostovi - prospektivna forenzička autopsijska studija

Introduction When the coronary artery, located subepicardially, submerges into the myocardium and appears again subepicardially after a short intramural course, it represents an embedded coronary artery, while the part of the myocardium above is a myocardial bridge. Objective We investigated the frequency of the embedded left coronary artery (LAD) in the autopsy material considering the descending branch of the LAD to be the most important one in the nourishment of the myocardium and myocardial bridges to be the most frequent in its area, as well as clinically important. Methods A prospective autopsy study of 975 cases was performed, including both, natural (21.33%) and violent (78.67%) deaths. The sample consisted of 74.56% males and 25.44% females. In order to discover myocardyal bridges and their characteristics, the hearts were examined by both transverse cuts and longitudinal openings of the LAD. Results Myocardial bridge was found in 78 cases (8.00%), more commonly in males (9.35%) than females (4.03%). The average length of the myocardial bridge was 21.85±16.10mm and thickness 3.744±1.48 mm. The common localization of the myocardial bridge was the proximal half of the LAD (89.74%). The upper part of the artery, proximal to the bridge, was a common site of atherosclerotic changes. Myocardial bridge was found in 12.50% of natural deaths, but in 13.38% out of all cases of sudden cardiac deaths. Conclusion Therefore, the presence of the myocardial bridge by itself is not predominant, but it is certainly a contributing factor to a sudden cardiac death.Uvod 'Poniruća koronarna arterija' je pojam koji se odnosi na slučaj kada koronarna arterija - inače lokalizovana subepikardno - ponire u miokard i ponovo se pojavljuje subepikardno, posle kratkog puta kroz mišić, pri čemu se mišić iznad nje naziva 'miokardni most'. Cilj rada Istraživali smo učestalost poniruće descendentne grane leve koronarne arterije u autopsijskom materijalu imajući u vidu da je ona najvažnija u ishrani srčanog mišića, da najčešće ponire u srčani mišić i da je vrlo značajna i s kliničkog aspekta. Metode rada Urađena je prospektivna autopsijska studija 975 slučajeva prirodnih (21,33%) i nasilnih smrti (78,67%). Uzorak je obuhvatio 74,56% osoba muškog i 25,44% ženskog pola. Radi otkrivanja miokardnog mosta i analize njegovih osobenosti, nishodna grana leve koronarne arterije otvarana je uzdužnim i poprečnim obdukcionim rezovima. Rezultati Miokardni most je utvrđen u 78 slučajeva (8,00%), češće kod muškaraca (9,35%) nego kod žena (4,03%). Prosečna dužina miokardnog mosta bila je 21,85±16,10 mm, a debljina 3,744±1,48 mm. Najčešća lokalizacija miokardnog mosta bila je u predelu proksimalne polovine descendentne grane leve koronarne arterije (89,74%), a aterosklerotske promene su najčešće ustanovljene ushodno od miokardnog mosta. Miokardni most je postojao u 12,50% slučajeva prirodnih smrti, odnosno u 13,38% slučajeva tzv. naprasnih srčanih smrti. Zaključak Postojanje miokardnog mosta nije predominantni činilac za pojavu naprasne srčane smrti, ali je njegovo postojanje faktor rizika za njen nastanak

Ameliorating effects of antioxidative compounds from four plant extracts in experimental models of diabetes

Given that oxidative stress plays a major role in pancreatic β-cell dysfunction and ultimate destruction, as well as in different complications of diabetes, therapy with antioxidants has assumed an important place in the management of diabetes. The relatively limited effects of established antioxidant compounds have stimulated efforts to develop new therapeutic strategies, e.g. to increase the endogenous antioxidant defences through pharmacological modulation of key antioxidant enzymes. Plant extracts are gaining popularity in treating diabetes because many substances synthesized by higher plants and fungi possess antioxidant activities and can prevent or protect tissues against the damaging effects of free radicals. This review summarizes experimental models of diabetes and possible mechanisms that lie behind the antioxidative effects of α-lipoic acid (LA), a powerful antioxidant and compound that stimulates cellular glucose uptake, as well as of plant extracts from sweet chestnut (Castanea sativa), edible mushroom (Lactarius deterrimus) and natural products containing β-glucans in the treatment of diabetes. Their roles in preventing pancreatic β-cell death and in ameliorating the effects of severe diabetic complications are discussed.Terapija antioksidansima zauzima značajno mesto u lečenju dijabetesa s obzirom da oksidativni stres u velikoj meri doprinosi narušavanju funkcije i strukture β-ćelija pankreasa kao i razvoju komplikacija u dijabetesu. Zbog ograničenog dejstva postojećih antioksidativnih jedinjenja traga se za novim terapijskim rešenjima u tretmanu dijabetesa, kao što je povećanje endogene antioksidativne zaštite organizma putem farmakološke modulacije ključnih antioksidativnih enzima. Primena biljnih ekstrakata u lečenju dijabetesa postaje sve popularnija. Mnoge supstance koje se nalaze u sastavu viših biljaka i gljiva poseduju antioksidativna svojstva koja mogu da zaštite tkiva od štetnih uticaja slobodnih radikala. U ovom revijalnom radu opisani su eksperimentalni modeli dijabetesa kao i mogući mehanizmi koji leže u osnovi antioksidativnog dejstva α-liponske kiseline (LA), snažnog antioksidansa i jedinjenja koje stimuliše ćelijsku apsorpciju glukoze, kao i biljnih ekstrakata izolovanih iz slatkog kestena (Castanea sativa), jestivih pečuraka (Lactarius deterrimus) i prirodnih proizvoda koji sadrže β-glukan u lečenju dijabetesa. Opisani su njihova uloga u sprečavanju smrti β-ćelija pankreasa kao i blagotvorno dejstvo na komplikacije u dijabetesu.Projekat ministarstva br. 17302

PARP-1 and YY1 Are Important Novel Regulators of CXCL12 Gene Transcription in Rat Pancreatic Beta Cells

Despite significant progress, the molecular mechanisms responsible for pancreatic beta cell depletion and development of diabetes remain poorly defined. At present, there is no preventive measure against diabetes. The positive impact of CXCL12 expression on the pancreatic beta cell prosurvival phenotype initiated this study. Our aim was to provide novel insight into the regulation of rat CXCL12 gene (Cxcl12) transcription. The roles of poly(ADP-ribose) polymerase-1 (PARP-1) and transcription factor Yin Yang 1 (YY1) in Cxcl12 transcription were studied by examining their in vitro and in vivo binding affinities for the Cxcl12 promoter in a pancreatic beta cell line by the electrophoretic mobility shift assay and chromatin immunoprecipitation. The regulatory activities of PARP-1 and YY1 were assessed in transfection experiments using a reporter vector with a Cxcl12 promoter sequence driving luciferase gene expression. Experimental evidence for PARP-1 and YY1 revealed their trans-acting potential, wherein PARP-1 displayed an inhibitory, and YY1 a strong activating effect on Cxcl12 transcription. Streptozotocin (STZ)-induced general toxicity in pancreatic beta cells was followed by changes in Cxcl12 promoter regulation. PARP-1 binding to the Cxcl12 promoter during basal and in STZ-compromised conditions led us to conclude that PARP-1 regulates constitutive Cxcl12 expression. During the early stage of oxidative stress, YY1 exhibited less affinity toward the Cxcl12 promoter while PARP-1 displayed strong binding. These interactions were accompanied by Cxcl12 downregulation. In the later stages of oxidative stress and intensive pancreatic beta cell injury, YY1 was highly expressed and firmly bound to Cxcl12 promoter in contrast to PARP-1. These interactions resulted in higher Cxcl12 expression. The observed ability of PARP-1 to downregulate, and of YY1 to upregulate Cxcl12 promoter activity anticipates corresponding effects in the natural context where the functional interplay of these proteins could finely balance Cxcl12 transcription

Digestomics of Japanese abalone in real food matrix

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 Abstract

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

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